Project Summary Nearly every aspect of neuronal function depends on the accurate polarization of membrane proteins to axons or dendrites. During the current award period we have identified the principal trafficking pathways that underlie the polarized localization of neuronal plasma membrane proteins in cultured hippocampal neurons and developed methods to image each of the different populations of the long-range carriers that transport proteins along these pathways. Our results show that the selectivity of anterograde, kinesin- mediated transport plays a central role in the targeting of polarized proteins. Carriers containing dendritic proteins are transported into dendrites but excluded from axons; carriers containing axonal proteins enter both dendrites and axons, but are transported preferentially into axons. We also developed an assay to assess the selective transport of constitutively active kinesin motor domains in the absence of cargo. We demonstrated that Kinesin-1 motor domains translocate preferentially into the axon, whereas a Kinesin-3 motor domain translocates with equal efficiency into both axons and dendrites. Recent evidence demonstrates that posttranslational modifications of tubulin (acetylation and glutamylation) regulate the efficiency of kinesin translocation and our preliminary data show that axonal and dendritic microtubules differ in both of these posttranslational modifications. Using the unique methods we have developed during the current award period, we now propose to identify the kinesins that transport each population of long-range carriers and to investigate the molecular features of kinesins and the molecular modifications of microtubules that determine the selectivity of carrier transport in hippocampal neurons. We will use two-color imaging to identify the carrier populations labeled by expressed kinesins. We will also use RNAi to inhibit the expression of individual kinesins and evaluate the populations of long-range carriers that are affected. We will compare the selectivity of motor domain translocation for all kinesin organelle motors expressed in hippocampal neurons and examine how posttranslational modifications of tubulin influence the selectivity of motor domain transport and the transport of long-range carriers. Defects in kinesin-mediated transport cause neuronal dysfunction in animal models and have been implicated in several human neurodegenerative diseases. Our results will identify novel targets for pharmacological manipulations that could compensate for transport defects and protect against neural degeneration. Project Narrative Nearly every aspect of neuronal function depends on the accurate transport and trafficking of membrane proteins; defects in the long-range transport of membrane proteins are thought to underlie several neurodegenerative diseases. By elucidating the regulatory mechanisms that underlie the accuracy of kinesin-driven transport, our work may identify a novel set of targets for pharmacologic manipulations that could enhance long-range transport and perhaps ameliorate neural degeneration.